Project Summary/Abstract One of the main reasons to study the bacterial mechanosensitive channel of large conductance, MscL, is that it has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction. With a crystal structure of what appears to be a ?nearly-closed? state of M. tuberculosis MscL, the channel has advanced the field considerably by allowing researchers to overlay genetic and molecular analyses, coupled with electrophysiology, onto a structural model. Thus, MscL from E. coli (Ec-MscL), which was the first defini- tive mechanosensitive channel identified, continues to serve as a tractable model for determining principles for how a protein senses and responds to membrane tension. However, another reason to study MscL is that the channel serves a vital function in maintaining osmotic homeostasis of microbes. It normally serves as a biolog- ical emergency release valve; upon osmotic downshock it opens a huge 30 pore that allows for the rapid re- lease of many accumulated cytoplasmic components, including potassium and glutamate, thus preventing cell lysis. When the channel gates inappropriately it can lead to the death of the microbial cell; it thus is a viable pharmacological target for potential antibiotics. Historically, one of the limitations in the study of MscL function and pharmacology has been the total lack of small molecular probes that bind and modulate the channel. From a High Throughput Screening (HTS) facility on campus, we have identified 18 novel chemical compounds that inhibit the growth of E. coli in a MscL-dependent manner; surprisingly, an additional hit was streptomycin, which appears to directly bind to and increase the probability of opening the MscL channel. We have used this system to develop and refine assays for determining compound efficacy. In 96 well plates we can assay cell growth to determine the minimal inhibitory concentrations (MIC) of compounds in the presence or absence of expressed Ec-MscL, MscL orthologues, or the unrelated bacterial mechanosensitive channel MscS as a nega- tive control. In addition, we have potassium and glutamate flux assays to measure the results of MscL gating in vivo, the ability to determine channel function by electrophysiology, and have developed assays to determine the binding sites of compounds. We will use this array of assays to determine if and how the novel compounds identified in the HTS bind and modulate MscL activity. These studies will yield insight into mechanosensitive channel gating mechanisms; in addition, co-crystallization of MscL with one or more of these compounds may yield an open state structure for MscL, and the findings could eventually lead to a new generation of antibiotics.